Bottom Line:
Δ21 IFITM3 also efficiently suppressed replication of H1N1 and, to a lesser extent, H3N2 IAV.Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than full-length IFITM3 but an abundant amount of both IFITM3 variants still localized to late endosomes and lysosomes.Our data indicate that tyrosine 20 partially regulates the subcellular localization of IFITM3 but is not functionally essential for IFITM3-mediated IAV restriction.

Affiliation: Department of Cell Biology and Neuroscience, College of Natural and Agricultural Sciences, University of California Riverside, Riverside, California, United States of America.

ABSTRACTThe IFITM3 polymorphism rs12252-C, which encodes an IFITM3 isoform (Δ21 IFITM3) lacking 21 amino acids at the amino terminus, has been controversially associated with poor clinical outcomes in patients with H1N1 influenza A virus (IAV) infections. In vitro studies have shown that Δ21 IFITM3 loses its ability to restrict H1N1 IAV. Subsequent research has also revealed that tyrosine 20 is the key determinant for IFITM3 endocytic trafficking, which is essential for the efficient anti-viral activity of IFITM3. In contrast to previous studies, we demonstrated that both Δ21 IFITM3 and an IFITM3 variant (Y20A IFITM3), in which tyrosine 20 is substituted with alanine, strongly restricted entry mediated by IAV H1, H3, H5, and H7 proteins. Δ21 IFITM3 also efficiently suppressed replication of H1N1 and, to a lesser extent, H3N2 IAV. Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than full-length IFITM3 but an abundant amount of both IFITM3 variants still localized to late endosomes and lysosomes. Our data indicate that tyrosine 20 partially regulates the subcellular localization of IFITM3 but is not functionally essential for IFITM3-mediated IAV restriction. They also suggested that mechanisms, other than viral entry restriction, might contribute to variations in clinical outcomes of H1N1 influenza associated with rs12252-C.

pone-0110096-g004: Δ21 IFITM3 and Y20A IFITM3 have boarder subcellular distributions.A549 cells expressing native FL IFITM3, Δ21 IFITM3, or Y20A IFITM3 were fixed, permeabilized, and labeled with anti-LAMP2 (green) and anti-IFITM2/3 (red) and DAPI (blue) antibodies. DAPI was used as nuclear counterstain. Cells were imaged by confocal microscopy. Each panel represents at least two sets of experiments with similar results.

Mentions:
The subcellular localization of IFITM proteins was thought to be critical for their inhibitory effects on viral entry. Jia et al. showed that— compared with flag-tagged FL IFITM3, which distributes mainly in late endosomes/lysosomes—flag-tagged Δ21 IFITM3 localizes primarily to the periphery of the cell [11]. To examine whether native IFITM3 isoforms have similar subcellular distributions, confocal analysis was performed. We observed that native Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than FL IFITM3. Nevertheless, a large amount of Δ21 IFITM3 and Y20A IFITM3 still colocalized with LAMP2, a late-endosomal/lysosomal marker (Fig. 4). When epitope tags were added to the N-terminus of IFITM3 isoforms, their subcellular distribution was changed. Both c-myc-tagged IFITM3 isoforms concentrated in late-endosomal/lysosomal compartments (Fig. 5A), whereas a minimal amount of flag-tagged FL IFITM3 and Δ21 IFITM3 colocalized with LAMP2 (Fig. 5B). These diverse patterns of subcellular distributions were compatible with our viral entry data. Flag-tagged Δ21 IFITM3, which mainly distributed to the plasma membrane, had the weakest effect on viral entry. However, endosome/lysosome-associated native Δ21 IFITM3 and Y20A IFITM3 might contribute to efficient inhibition of IAV infection. The broader distributions of Δ21 IFITM3 and Y20A IFITM3 (compared with FL IFITM3) at both the plasma and endosomal/lysosomal membranes raises the possibility that the N-terminal 21 amino acids and tyrosine 20 play important roles but may not be the only determinants contributing to the subcellular localization of IFITM3. Because epitope tags altered the subcellular distribution and interfered with the function of IFITM3, native IFITM3 isoforms would be a better model for IFITM3-related studies.

pone-0110096-g004: Δ21 IFITM3 and Y20A IFITM3 have boarder subcellular distributions.A549 cells expressing native FL IFITM3, Δ21 IFITM3, or Y20A IFITM3 were fixed, permeabilized, and labeled with anti-LAMP2 (green) and anti-IFITM2/3 (red) and DAPI (blue) antibodies. DAPI was used as nuclear counterstain. Cells were imaged by confocal microscopy. Each panel represents at least two sets of experiments with similar results.

Mentions:
The subcellular localization of IFITM proteins was thought to be critical for their inhibitory effects on viral entry. Jia et al. showed that— compared with flag-tagged FL IFITM3, which distributes mainly in late endosomes/lysosomes—flag-tagged Δ21 IFITM3 localizes primarily to the periphery of the cell [11]. To examine whether native IFITM3 isoforms have similar subcellular distributions, confocal analysis was performed. We observed that native Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than FL IFITM3. Nevertheless, a large amount of Δ21 IFITM3 and Y20A IFITM3 still colocalized with LAMP2, a late-endosomal/lysosomal marker (Fig. 4). When epitope tags were added to the N-terminus of IFITM3 isoforms, their subcellular distribution was changed. Both c-myc-tagged IFITM3 isoforms concentrated in late-endosomal/lysosomal compartments (Fig. 5A), whereas a minimal amount of flag-tagged FL IFITM3 and Δ21 IFITM3 colocalized with LAMP2 (Fig. 5B). These diverse patterns of subcellular distributions were compatible with our viral entry data. Flag-tagged Δ21 IFITM3, which mainly distributed to the plasma membrane, had the weakest effect on viral entry. However, endosome/lysosome-associated native Δ21 IFITM3 and Y20A IFITM3 might contribute to efficient inhibition of IAV infection. The broader distributions of Δ21 IFITM3 and Y20A IFITM3 (compared with FL IFITM3) at both the plasma and endosomal/lysosomal membranes raises the possibility that the N-terminal 21 amino acids and tyrosine 20 play important roles but may not be the only determinants contributing to the subcellular localization of IFITM3. Because epitope tags altered the subcellular distribution and interfered with the function of IFITM3, native IFITM3 isoforms would be a better model for IFITM3-related studies.

Bottom Line:
Δ21 IFITM3 also efficiently suppressed replication of H1N1 and, to a lesser extent, H3N2 IAV.Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than full-length IFITM3 but an abundant amount of both IFITM3 variants still localized to late endosomes and lysosomes.Our data indicate that tyrosine 20 partially regulates the subcellular localization of IFITM3 but is not functionally essential for IFITM3-mediated IAV restriction.

Affiliation:
Department of Cell Biology and Neuroscience, College of Natural and Agricultural Sciences, University of California Riverside, Riverside, California, United States of America.

ABSTRACTThe IFITM3 polymorphism rs12252-C, which encodes an IFITM3 isoform (Δ21 IFITM3) lacking 21 amino acids at the amino terminus, has been controversially associated with poor clinical outcomes in patients with H1N1 influenza A virus (IAV) infections. In vitro studies have shown that Δ21 IFITM3 loses its ability to restrict H1N1 IAV. Subsequent research has also revealed that tyrosine 20 is the key determinant for IFITM3 endocytic trafficking, which is essential for the efficient anti-viral activity of IFITM3. In contrast to previous studies, we demonstrated that both Δ21 IFITM3 and an IFITM3 variant (Y20A IFITM3), in which tyrosine 20 is substituted with alanine, strongly restricted entry mediated by IAV H1, H3, H5, and H7 proteins. Δ21 IFITM3 also efficiently suppressed replication of H1N1 and, to a lesser extent, H3N2 IAV. Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than full-length IFITM3 but an abundant amount of both IFITM3 variants still localized to late endosomes and lysosomes. Our data indicate that tyrosine 20 partially regulates the subcellular localization of IFITM3 but is not functionally essential for IFITM3-mediated IAV restriction. They also suggested that mechanisms, other than viral entry restriction, might contribute to variations in clinical outcomes of H1N1 influenza associated with rs12252-C.